Multimodal nanoparticles (NPs) advantageously possess properties such as a size similar to those of proteins or nucleic acids, large interactions due to their high surface area to volume ratio, complementary combination of active units within a single platform, high active material localization, structural diversity, and long circulation time in blood compared to small molecules.
The elaboration of bimodal nanoparticles, combining fluorescence and magnetism, has fostered great interest for their promising potential in dual bioimaging for diagnosis, remote drug vectorization, therapy by hyperthermia and selection of biological entities. While fluorescence detection is characterized by high sensitivity, addressing magnetic properties opens up the possibility of deep tissue imaging and remote mass transfer.
Most of the common systems that have been elaborated so far consist of iron oxide nanoparticles (magnetic), which are known for their relative innocuity, coated with organic or inorganic functional units (fluorescent). Alternative doped or core-shell structures have been elaborated with luminescent lanthanides or quantum dots, and yet these latter structures require prior encapsulation in silica or latex matrices to be protected from emission quenching by the surroundings. (Cheon and Lee, Acc. Chem. Res. 2008, 41, 1630-1640; Lee et al., Chem. Soc. Rev. 2012, 41, 2575-2589; Fan et al., ACS Nano 2012, 6, 1065-1073; Bigall et al., Nano Today, 2012, 7, 282-296).
For all these systems, deleterious electron transfer effects exerted by the magnetic nanoparticles on the luminophore emission have been noted, which is reinforced by the usually low payload of luminophores compared to that of iron oxide nanoparticles.
Moreover, these nanoparticles suffer from the following drawbacks:                leakage or disassembling of the fluorescent molecules or the magnetic nanoparticles, especially in biological media, leading to a loss of combined fluorescence and magnetism, a decrease in the performances, and the appearance of noise signal;        low payload of fluorescent and magnetic active units, compelling to use high doses;        high photobleaching yield in the case of individual organic fluorescent molecules;        high sensitivity of fluorescence towards the biological surroundings causing possible emission quenching or color shift;        weak size discrimination leading to non-specific targeting of organs and/or malignant cells;        low half-life in biological media, especially short circulation time in blood causing rapid clearance of materials following intravenous injection;        difficulties to further functionalize the nanoparticle surface, whereas functionalization brings strong added value in terms of biological targeting and action.        
Surprisingly, core-shell structures, based on an organic emissive platform (fluorescent) surrounded by magnetic nanoparticles have never been proposed to address these issues.
The present invention thus relates to nanoassemblies comprising a matrix-free organic fluorescent core surrounded by magnetic nanoparticles. The resulting reverse architecture of the nanoassemblies of the invention resembles a “raspberry like” arrangement (FIG. 1) (Faucon et al., J. Mater. Chem. C., 2013, 1, 3879-3886). In the following, such reverse architectures are referred to as “fluo@mag” nanoassemblies.
The Applicant evidenced that such reverse architectures provide dense emissive core where the embedded fluorescent molecules become insensitive to the pH or the ionic strength of the media, and provide stable fluorescence signal under usual light observation conditions. Moreover, emission quenching from the magnetic nanoparticles through deleterious electron transfer is severely reduced due to effective separation of the magnetic nanoparticles from the fluorescent core. More than 70% of the core's fluorescence is retained after addition of the magnetic nanoparticles. Positioning magnetic nanoparticles on the periphery creates a charged shell structure which acts as a repelling electrostatic barrier to ions or hydrophobic molecules. As a result, fluorescent molecules are protected from possible dissolution in hydrophobic media or action by enzymes, which would result in the disassembling/release of intact or transformed individual fluorescent molecules. This in turn reinforces the fluorescent core cohesion through strong interactions of the fluorescent molecules, hence destabilization upon solvation is unlikely to occur. By contrast, the possible loss of magnetic nanoparticles would cause little effects in terms of spurious signal as the magnetic response of individual nanoparticles is smaller than that of the final bimodal assemblies.
The fluo@mag nanoassemblies of the invention present many advantages as described above. Due to their manufacturing process, illustrated in the experimental part below, they are obtained and stabilized under dilute acidic conditions, preferably using nitric acid. These acidic conditions are not optimized for biological purposes, especially for in vivo imaging. There is thus a need for stabilization of the nanoassemblies of the invention in water and physiological conditions.
Moreover, it would be interesting to functionalize the nanoassemblies of the invention by bioactive molecules in order to widen the range of possible biological applications of the nanoassemblies. Care should be taken that functionalization of the nanoassemblies does not lead to the aggregation of nanoassemblies.
The Applicant evidenced that stabilization in physiological conditions and functionalization may be achieved by the addition of a polymeric protective shell around the nanoassembly of the invention.
Especially, the Applicant showed that coating the surface of the nanoassembly of the invention using a polymer may achieve the above expected effects (FIG. 2). In the following, such fluo@mag nanoassemblies coated with a polymer are referred to as fluo@ mag@polymer nanoassemblies.
The polymer used to modify the nanoassemblies of the invention should meet the following specifications:                Interactions between the polymer and the nanoassembly should provide overall stiff cohesion to the fluo@mag@polymer nanoassembly. For that purpose, electrostatic interactions with the positively charged surface of the nanoassemblies are preferred. Moreover, the polymer preferably comprises a multiplicity of sites interacting with the nanoassembly.        The association constant between the polymer and the magnetic nanoparticles should be lower than the association constant between the fluorescent core and the magnetic nanoparticles. In other words, the polymer should have enough affinity for the magnetic nanoparticles present at the surface of the fluorescent core in order to coat the nanoassembly, while avoiding to take off part of the magnetic shell.        The presence of the polymer should not induce the aggregation of the nanoassemblies both during manufacturing and during use in biological media.        The polymer should be functionalizable, preferably the polymer should present a multiplicity of functionalizable patterns.        The polymer should present biocompatibility.        
The Applicant evidenced that negatively charged polyelectrolytes meet above specifications. According to a specific embodiment of the invention, the preferred polymer is polyacrylic acid (PAA).
Advantageously, the mean size of the fluo@mag@polymer does increase in a reasonable range compared to that of the uncoated fluo@mag nanoassembly, keeping the adequate dimensions for applications in vivo.